For the reporter constructs, the VEGF-A promoter regions (−2,000 to +50 bp) were amplified from human genomic DNA (Zyagen, San Diego, CA, USA) by PCR using iProof™ High-Fidelity DNA Polymerase (Bio-Rad Laboratories, Inc.) (23 (link)). The PCR products were subcloned into the pGL4.11[luc2P] vector (Promega Corporation, Madison, WI, USA) upstream of a luciferase gene using appropriate restriction sites. Putative transcription factor binding sites were identified with the Transcription Element Search System (TESS) algorithm (http://www.cbil.upenn.edu/downloads/ ).
The transcription factor binding to the VEGF-A promoter was analyzed by luciferase assay as previously reported (20 (link),23 (link)). Briefly, MCF-7 cells (1×105) were transfected in 24-well plates with 1 µg pGL4.11[luc2P] luciferase reporter vector driven by the VEGF-A promoter, or with 2 µg control vector. For transfection of MCF-7 cells, 0.2×105 cells/well were seeded into a 24-well plate and grown for 24–48 h. Cells were harvested 48 h post-transfection, and efficiency was measured by qPCR, immunostaining and western blotting.
The transcription factor binding to the VEGF-A promoter was analyzed by luciferase assay as previously reported (20 (link),23 (link)). Briefly, MCF-7 cells (1×105) were transfected in 24-well plates with 1 µg pGL4.11[luc2P] luciferase reporter vector driven by the VEGF-A promoter, or with 2 µg control vector. For transfection of MCF-7 cells, 0.2×105 cells/well were seeded into a 24-well plate and grown for 24–48 h. Cells were harvested 48 h post-transfection, and efficiency was measured by qPCR, immunostaining and western blotting.
